microscopy

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This is a series of lectures on microbiology useful for undergraduate medical and paramedical students

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    1. 1. Microscopy Dr. Ashish Jawarkar M.D. Consultant Pathologist Parul Sevashram Hospital
    2. 2. Types of Microscopes: 1. Compound Light Microscope (what we use most often) 2. Stereoscopes – also known as dissecting scopes 3. Electron Microscopes Dr. Ashish V. Jawarkar
    3. 3. Parts of the Microscope Arm Dr. Ashish V. Jawarkar
    4. 4. Parts of the Microscope Diaphragm Light Source Dr. Ashish V. Jawarkar
    5. 5. Parts of the Microscope Stage Stage Clips Dr. Ashish V. Jawarkar
    6. 6. Parts of the Microscope Revolving Nosepiece Objective Lenses Dr. Ashish V. Jawarkar
    7. 7. Parts of the Microscope Ocular Lens Dr. Ashish V. Jawarkar
    8. 8. Parts of the Microscope Coarse adjustment knob Used only when low power objective is used!! Dr. Ashish V. Jawarkar
    9. 9. Parts of the Microscope Fine adjustment knob Dr. Ashish V. Jawarkar
    10. 10. Important Vocabulary : magnification  mag-ne-fe-'ka-shen n 1. apparent enlargement of an object 2. the ratio of image size to actual size A magnification of "100x" means that the image is 100 times bigger than the actual object. resolution  rez-e-loo-shen n 1. clarity, sharpness 2. the ability of a microscope to show two very close points separately Dr. Ashish V. Jawarkar
    11. 11. Carrying a Microscope Dr. Ashish V. Jawarkar
    12. 12. Parts of the Microscope Arm Dr. Ashish V. Jawarkar
    13. 13. Steps to Use: 1. Rotate the low power objective into place and make sure the stage is all the way down. 2. Place slide on stage making sure object to be viewed is centered over the hole in the stage. Use the stage clips to hold the slide in place. 3. Turn light on. 4. Focus first with the coarse adjustment knob. Once in focus on low power, turn the nosepiece until the next higher lens is in place. 5. Use FINE adjustment knob ONLY and focus the object. Dr. Ashish V. Jawarkar
    14. 14. Techniques of Light Microscopy • Preparation of Specimens for the Light Microscope: • 1) Wet Mounts- drop of medium with microbes is spread on a slide • 2) Smears- microbes from a loopful of medium are spread on a slide, then heat fixed to kill microbes - heat fixationDr. Ashish V. Jawarkar
    15. 15. Making a wet mount: Dr. Ashish V. Jawarkar
    16. 16. Wet Mounts: Poorly Done: Nicely Done: Dr. Ashish V. Jawarkar
    17. 17. Principles of Staining • Stain- dye that binds to a cellular structure and gives it color • + charge-basic= methylene blue, crystal violet, safranin and malachite green • - charge-acidic= eosin and picric acid • Simple stain- single dye and reveals basic cell shapes and structures • Differential stain- 2 or more dyes: Gram stain, Ziehl-Neelsen acid fast and spore Dr. Ashish V. Jawarkar
    18. 18. Gram Stain • Gram Stain- 1884 crystal violet (+) and iodine and ethanol decolorizer, and counterstained with safranin (-) • Gram +=purple • Gram - = red • Gram non reactive= no stain • Gram Variable= stain unevenly Dr. Ashish V. Jawarkar
    19. 19. Special Staining Procedures • Ziehl-Neelsen Acid-Fast Stain - 1882 modification of Ehrlich staining method - Acid fast retain red color in cell walls • Negative staining-capsule is present and won’t take up stain • Flagellar staining- coats flagella so they can be seen • Endospore staining- Schaeffer-Fulton stain Dr. Ashish V. Jawarkar
    20. 20. Recording what you see: Include: 1. Figure #: and Title 2. Labeled drawing of the field of view. Label on the right using straight lines which should never cross. 3. Common and scientific name of organism. 4. Magnification you were viewing when you drew the organism: ocular X objective Dr. Ashish V. Jawarkar
    21. 21. Remember: 1. If you are seeing perfectly round, clear circles then you just may be looking at air bubbles. Check your slide and try again. 2. Microscopes must always be properly put away. 3. Slides and cover-slips should be washed, dried, and returned to their proper place. Dr. Ashish V. Jawarkar

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